P481: MP0533, A NEW MULTISPECIFIC DARPIN CD3 ENGAGER TARGETING THREE TUMOR ASSOCIATED ANTIGENS, INDUCES SPECIFIC T-CELL ACTIVATION AND AML TUMOR KILLING IN VIVO
Authors: A. Croset, M. Bianchi, M. Franchini, T. Lekishvili, Y. Kaufmann, A. Auge, M. Hänggi, W. Ali, C. Zitt, S. Wullschleger, M. Matzner, C. Reichen, S. Fischer, Y. Grübler, A. Eggenschwiler, T. Looser, R. Watson, P. Spitzli, V. Kirkin, D. Steiner, A. Goubier
Journal: HemaSphere (2022)

Background: AML treatment options are generally focused on chemotherapy followed by allogeneic hematopoietic stem cell transplantation. However, many patients are ineligible for these options and often receive palliative treatments with poor long-term survival and there is an urgent need for improved therapeutic solutions. Newer therapies, including T cell engagers (TCE) and chimeric antigen receptor (CAR) T cells that target specific molecules overexpressed on leukemic blasts and stem cells are promising alternative treatment options for AML. However, achieving efficacy and low toxicity remains challenging. MP0533 is a multi-specific, half-life extended, T cell engaging DARPin in development for treatment of patients with AML and higher risk myelodysplastic syndrome (HR-MDS). MP0533 consists of a chain of 6 covalently linked DARPin domains. Three of these domains engage CD33, CD123 and CD70 on the target cells, utilizing an avidity driven approach to ensure that binding occurs preferentially when 2 or more TAAs are present. A fourth DARPin domain targets CD3 for T-cell engagement. Lastly 2 additional HSA binding DARPin domains prolong half-life in circulation. In vitro, MP0533 has demonstrated, in both allogeneic and autologous settings, single to double digit pM potency against AML cell lines and AML primary cells expressing any combination of at least 2 of the 3 targeted TAAs and low cytokine release. Aims: To evaluate and confirm that the previously established in vitro and ex vivo efficacy and safety profiles of MP0533 are also observed in an in vivo human PBMC xenograft mouse model. Methods: NXG mice were injected intraperitoneally with hPBMC from healthy donors and xenografted with MOLM-13 cells subcutaneously two days after hPBMC injection. Therapeutic treatment was initiated eight days after tumor implantation once tumors were established. DARPins were injected intravenously three times per week for two weeks. Several readouts such as T cell activation and human immune cell infiltration in tumors were performed by flow cytometry three days after first treatment. Additionally, cytokine and chemokine release were analyzed in serum four hours after the first treatment injection and in tumor supernatant 3 days after the first treatment injection. Results: MP0533 induced AML tumor killing in vivo (P value <0.0001 at day 18 compared to PBS). Three days after the first injection, MP0533 recruited human immune cells in the tumor (around 10% of hCD45+ cells detected by FACS, P value = 0.0044 compared to PBS) and induced T cell activation (around 46% of CD4+/CD25+/CD69+ cells and 38% of CD8+/CD25+/CD69+ cells detected by flow cytometry, P value <0.0001 compared to PBS) which triggered cytokines and chemokines release in the tumor such as IL-6, INFγ and TNFα. However, MP0533 didn’t induce significant cytokine release, including at the highest tested dose, in mice serum four hours after the first injection. Summary/Conclusion: We were able to generate a multi-specific CD3 engaging DARPin molecule with tailored affinities towards different TAAs showing significant efficacy in vivo that induced T cell activation without significant cytokine/chemokine release.